Natural Killer T (NKT) cells are an evolutionarily conserved subset of innate-like T cells that recognizes lipid antigens presented by CD1d molecules. NKT cells have been shown to play critical roles in autoimmunity, and in immunity to cancer and infection. In addition to the widely studied invariant NKT (iNKT) cells that utilize V14-J18 TCR-chains and recognize the prototypical -galactosylceramide antigen, CD1d-restricted NKT cells utilizing diverse TCRs (dNKT or type II NKT cells) exist. Available data suggest that dNKT cells have antigen specificities and functional capabilities that are distinct from those of iNKT cells. However, it is not known whether dNKT cells recognize microbial lipid antigens, whether they have the ability to generate memory responses, and what their function is during infection. Progress in understanding these critical questions is in large part hindered b the lack of markers, antigens, and tools to specifically identify and study dNKT cells. Our long-term goal is to understand the antigen specificity of dNKT cells, their function in response to microbial stimulation, and their role during Mtb infection. The objective here, which is the next step in pursuit of that goal, is to generate and characterize a TCR-retrogenic mouse model for the in vivo study of dNKT cells. Based on the previously unrecognized ability of dNKT cells to recognize diverse mycobacterial lipid antigens and to be stimulated by Mtb-infected APCs, our central hypothesis is that dNKT cells have a protective function during Mtb infection. Our strategy is to generate and characterize retrogenic (Rg) mice expressing the TCRs used by Mtb-specific dNKT cells. Generation of Rg mice is a novel method for rapid expression of defined TCR and proteins that uses retroviral and stem cell-mediated gene transfer. We have used this method successfully to generate four Rg mouse lines that express MHC class I- restricted TCRs from Mtb-specific CD8+ T cells. We plan to test our central hypothesis and, thereby, accomplish the objective of this application by generating and characterizing TCR-retrogenic mice expressing CD1d-restricted Mtb-specific dNKT cell TCRs (Aim 1). We will then characterize Mtb-specific dNKT cell responses in those retrogenic mice following antigen stimulation and Mtb infection in vitro and in vivo (Aim 2). With respect to expected outcomes, the proposed studies will generate a murine in vivo system to enable studies of dNKT cell function in response to Mtb antigens and during Mtb infection. These studies are expected to have a significant impact because they will develop the first tractable small animal model for the study of dNKT cells in vivo, a model that can be used to study the contribution of dNKT cells to a variety of disease processes. Second, we expect to define the role of dNKT cells during mycobacterial infection, which may result in the identification of new antigen targets that could be incorporated into vaccines against Mtb. This work will fundamentally advance the field of CD1d/dNKT cell biology.